Adenoviruses
Among the most commonly used vectors for the delivery of genetic material into human cells are the Adenoviruses. Adenoviruses have been isolated from a large number of different species, and more than 100 different serotypes have been reported. The overall organization of the Adenoviral genome is conserved among serotypes, such that specific functions are similarly positioned. The Ad2 and Ad5 genomes have been completely sequenced and sequences of selected regions of genomes from other serotypes are available. Most adults have been exposed to the Adenovirus serotypes most commonly used in gene therapy (serotypes 2 and 5).
The Ad5 genome is a linear, non-segmented, double stranded DNA, approximately 34-43 kbp (size varies from group to group) which has the theoretical capacity to encode 30-40 genes. The Ad5 genome is flanked on both sides by inverted terminal repeat sequences (LITR and RITR), which are essential to replication of Adenoviruses. The virus infectious cycle is divided into an early and a late phase. In the early phase, the virus is uncoated and the genome transported to the nucleus, after which the early gene regions E1-E4 become transcriptionally active.
The early region-1 (E1) contains two transcription regions named E1A and E1B. The E1A region (sometimes referred to as immediate early region) encodes two major proteins that are involved in modification of the host-cell cycle and activation of the other viral transcription regions. The E1B region encodes two major proteins, 19K and 55K, that prevent, via different routes, the induction of apoptosis resulting from the activity of the E1A proteins. In addition, the E1B-55K protein is required in the late phase for selective viral mRNA transport and inhibition of host protein expression. Early region-2 (E2) is also divided into an E2A and E2B region that together encode three proteins, DNA binding protein, viral polymerase and pre-terminal protein, all involved in replication of the viral genome. The E3 region is not necessary for replication in vitro but encodes several proteins that subvert the host defense mechanism towards viral infection. The E4 region encodes at least six proteins involved in several distinct functions related to viral mRNA splicing and transport, host-cell mRNA transport, viral and cellular transcription and transformation.
The late proteins necessary for formation of the viral capsids and packaging of viral genomes, are all generated from the major late transcription unit (MLTU) that becomes fully active after the onset of viral DNA replication. A complex process of differential splicing and polyadenylation gives rise to more than 15 mRNA species that share a tripartite leader sequence. The early proteins E1B-55K and E4-Orf3 and Orf6 play a pivotal role in the regulation of late viral mRNA processing and transport from the nucleus.
Packaging of newly formed viral genomes in pre-formed capsids is mediated by at least two Adenoviral proteins, the late protein 52/55K and an intermediate protein IVa2, through interaction with the viral packaging signal (Ψ) located at the left end of the Ad5 genome. A second intermediate protein, pIX, is part of the capsid and is known to stabilize the hexon-hexon interactions. In addition, pIX has been described to transactivate TATA-containing promoters like the E1A promoter and the major late promoter (MLP).
Adenovirus-Based Vectors and Adenoviral Packaging Cell Lines
Adenovirus-based vectors have been used as a means to achieve high-level gene transfer into various cell types, as vaccine delivery vehicles, for gene transfer into allogeneic tissue transplants for gene therapy, and to express recombinant proteins in cell lines and tissues that are otherwise difficult to transfect with high efficiency. The current known systems for packaging Adenovirus-based vectors consist of a host cell and a source of the Adenoviral late genes. The current known host cell lines, including the 293, QBI, and PERC 6 cells, express only early (non-structural) Adenovirus (Ad) genes, not the Ad late (structural) genes needed for packaging. The Ad late genes have previously been provided either by the Ad vectors themselves or by a helper Ad virus. Recently, “gutless” Adenoviral vectors—vectors that are devoid of all viral-protein-coding DNA sequences—have been developed. The gutless Adenoviral vectors contain only the ends of the viral genome (LITR and RITR), therapeutic gene sequences, and the normal packaging recognition signal (Ψ), which allows this genome to be selectively packaged and released from cells. However, to propogate the gutless adenoviral vector requires a helper adenovirus (the helper) that contains the adenoviral genes required for replication and virion assembly as well as LITR, RITR, and Ψ. While this helper-dependent system allows the introduction of up to about 32 kb of foreign DNA, the helper virus contaminates the preparations of gutless Adenoviral vectors. This contaminating replication competent helper virus poses serious problems for gene therapy, vaccine, and transplant applications both because of the replication competent virus and because of the host's immune response to the adenoviral genes in the helper virus. One approach to decrease helper contamination in this helper-dependent vector system, has been to introduce a conditional gene defect in the packaging recognition signal (Ψ) making it less likely that its DNA is packaged into a virion. Gutless Adenoviral vectors produced in such systems still have significant contamination with helper virus. Being able to produce gutless Adenoviral gene transfer vectors without helper virus contamination would offer further reduced toxicity and prolonged gene expression in animals.
It is believed that these Ad late genes in the vector or in the helper Ad virus: 1) contribute to the inflammatory response to the Adenovirus vector in gene therapy applications, 2) interfere with the immune response in vaccine applications, 3) induce immune non-responsiveness to Adenovirus in allogeneic transplant applications, and 4) result in protein contaminants in protein expression applications. Further, they occupy space in the Adenoviral vector that could beneficially be used for carrying other genetic information. Remarkable progress has been made with these vectors in the last decade, but some shortcomings continue to challenge investigators.
Adenovirus Vectors for Gene Therapy and Protein Expression
Gene delivery or gene therapy is a promising method for the treatment of acquired and inherited diseases. An ever-expanding array of genes for which abnormal expression is associated with life-threatening human diseases are being cloned and identified. The ability to express such cloned genes in humans will ultimately permit the prevention and/or cure of many important human diseases, diseases for which current therapies are either inadequate or non-existent. Unfortunately, however, gene therapy protocols described to date have been plagued by a variety of problems, including in particular the short period of gene expression from the vector and the inability to effectively readminister the same vector a second time, both of which are caused by the host immune response against antigens associated with the vector and its therapeutic payload. Tissues that have incorporated the viral and/or therapeutic genes are initially attacked by the host's cellular immune response, mediated by CD8+ cytotoxic T cells as well as CD4+ helper T cells, which dramatically limits the persistence of gene expression from the vectors. Moreover, the host's humoral immune response mediated by the CD4+ T cells further limits the effectiveness of current gene therapy protocols by inhibiting the successful readministration of the same vector.
For example, following an initial administration of an Adenoviral vector, serotype-specific antibodies are generated against epitopes of the major viral capsid proteins, namely the penton, hexon and fiber. Given that such capsid proteins are the means by which the Adenovirus attaches itself to a cell and subsequently infects the cell, such antibodies are then able to block or “neutralize” reinfection of a cell by the same serotype of Adenovirus. This necessitates using a different serotype of Adenovirus in order to administer one or more subsequent doses of exogenous therapeutic DNA in the context of gene therapy and vaccines. In addition, both therapeutic and viral gene products are expressed on the target cells making them susceptible to cellular immune responses. Thus, they are rejected and the beneficial effect of the gene therapy is negated and the target organ or tissue may be destroyed. As a result of these immune-related obstacles, progress in gene therapy protocols has been stymied.
A large research effort has been mounted to optimize virus-based gene transfer vectors. Yet, the initial promise of gene therapy has been undermined by the biology of the commonly used viral vectors. For example, retroviruses are intrinsically mutagenic and oncogenic as they integrate into the human genome, and currently available Adenoviral vectors induce vigorous humoral and cellular immune responses that negate their therapeutic potential. Although the mutagenic and oncogenic properties of retroviral vectors are intrinsic, the immunogenicity of Adenoviral vectors may be mitigated. These responses arise from Adenoviral gene products expressed from the Adenoviral vector itself or from associated helper virus.
Adenovirus Vectors for Immunosuppressive Therapy
Transplants of allogeneic cells and tissues are an increasingly frequent and important method of treating various disease and conditions. With the advent of embryonic and other stem cell based therapies, there will be a further increase in such transplants. One challenge faced by such transplants is rejection by the recipient's immune system. Such rejection is prevented by long-term treatment with general immune suppressants such as rapamycin and cyclosporine A. However, treatment with such general immune suppressants results in an inhibition of protective immunity, resulting in susceptibility to a host of bacterial, viral, and fungal infections with associated morbidity and mortality.
A number of methods have been proposed for inducing specific immune suppression directed at the allogeneic cells or tissues transplanted. One of these methods is based on the classical “veto effect” that employs donor-derived CD8+ T cells to inhibit cellular immune responses. Yet, allogeneic grafts may only be partially protected by classical veto as CD8+ T cells may fail to remove organ-specific allo-reactive T cells.
Inducing the veto effect can be accomplished by a number of methods that result in the presence of CD8 on the surface of the transplanted allogeneic cells, including treating the allogeneic transplant with a protein fusion of CD8 and an antibody specific for a protein present of the allogeneic cells or tissues to be transplanted. The CD8 can also be “engineered” to the surface of the cells by introducing a transcriptionally and translationally active copy of the CD8 gene to the cells or tissues to be transplanted.
Adenoviral vectors are particularly suited for transduction of the CD8 gene to allogeneic cells/tissues for transplantation because they infect a wide range of cell and tissues with high efficiency and because the transduced DNA is expressed transiently and not permanently integrated into the genome of the transduced cells.
The ability of the CD8 to induce long-term immune non-responsiveness raises a challenge for the use of Adenoviral vectors: the expression of Adenoviral genes in conjunction with the CD8 gene form cells in the allogeneic transplant may induce the transplant recipient to a state of long-term non-responsiveness to the Adenovirus used as the basis for the vector. Adenovirus is a human pathogen and though not normally a great risk, it is associated with significant morbidity in immunocompromised people such as AIDS patients incapable of mounting an immune response to it.
At least 53 different forms of human Adenovirus have been characterized. The discriminating factor among these viruses is the humoral immune (i.e. antibody) response to the capsid hexon protein (encoded by various alleles of the L3 gene). In fact, the majority of variation among the different hexon proteins occurs in three “hyper”-variable regions; the humoral immune response to Adenoviruses is centered on these hypervariable regions.
The use of Adenovirus to deliver CD8 to protect allogeneic transplants from rejection poses unique problems, not the same as posed by other uses of Adenoviral vectors for more standard gene therapy protocols. Specifically, in standard gene therapy, the injection of a large number of Adenovirus particles into the patient may activate the pre-existing immunity to the Adenoviral vector which can interfere with the transduction of the therapeutic gene, lead to inflammatory responses, and in extreme cases the immune response result in death of the patient. The source of Adenoviral antigens engaging the pre-existing immunity can come both from the gene therapy virions and from newly synthesized Adenoviral proteins produced by infected cells.
Two advances have sought to overcome these problems are the use of “gutless” (fully-deleted) Adenoviral vectors and the use of rare Adenoviral hexons. While the use of “gutless” Adenoviral vectors removes the L3 gene from the therapeutic vector, the propagation of these “gutless” viruses requires the presence of helper Adenovirus that still contains L3 genes. And these helper viruses are significant contaminants in the therapeutic preparations of the “gutless” Adenoviral vectors. The use of L3 genes from rare adenoserotypes may avoid the problem of pre-existing immunity in that fraction of patients who have not been previously exposed to the Adenoviral serotype. Still, as the Adenoviral hexon proteins are highly immunogenic, there is a high probability that repeated treatments with an Adenoviral gene delivery vector based on a rare serotype will eventually induce an immune reaction, including neutralizing antibodies. In summary, the problem with Adenoviral vectors for classical gene therapy protocols is the presence or development of an immune response to the Adenoviral proteins that interferes with transduction of the therapeutic gene and/or causes inflammatory or other immune responses.
In the use of Adenovirus to deliver the CD8 gene to allogeneic cells/tissues ex vivo before reimplantation, the problem of immune reaction to the Adenoviral genes is quite different: here one is concerned with the induction of long-term immune non-responsiveness to whichever Adenovirus serotype serves as the basis for the vector.
Adenoviruses as Vaccine Vectors
Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors as they induce both innate and adaptive immune responses in mammalian hosts. Currently, Adenovirus vectors are being tested as subunit vaccine systems for numerous infectious agents ranging from malaria to HIV-1. Additionally, they are being explored as vaccines against a multitude of tumor-associated antigens. Thus far, most efforts have focused on vectors derived from Adenovirus of the human serotype 5 (AdHu5) for eventual use as vaccines for humans, while bovine, porcine, and ovine Adenoviruses have been explored for veterinary use.
The dynamics of Adenoviral gene expression have made the production of true Adenoviral packaging cell lines difficult: expression of the Adenoviral early functional transcription region (E1A) gene induces expression of the Adenoviral late genes (structural, immunogenic genes), which in turn kills the cell. Accordingly, a host cell that constitutively expresses the Adenoviral early genes cannot carry the “wild-type” Adenoviral late cistron. Previous host cells for propagating Adenoviral vectors are not “packaging” cells. Specifically, the 293, QBI and PERC 6 cells express only early (non-structural) Adenoviral genes, not the Adenoviral late genes needed for packaging. The Adenoviral late genes have previously been provided either by the Adenoviral vector or by a helper Adenoviral virus. These Adenoviral late genes in the Adenoviral vector or in a helper Adenoviral virus contribute to the inflammatory response to the Adenoviral vector; interfere with the immune response to Adenoviral based vaccines; induce immune non-responsiveness to Adenovirus in allogeneic transplant applications, and contribute to contamination in Adenoviral based protein expression. Further, they occupy space that could beneficially be used for carrying other genetic information.
The described invention addresses this problem and provides systems and methods for the construction of fully-deleted helper-independent Adenoviral vectors and uses thereof.